The insulin-like growth factors evolved in lower animalsto enable a wide range of physiological processes including smell, food consumption, metabolism, growth, reproduction and dormancy. These functions were accomplished by the actions of multiple related ligands that activated a common transmembrane receptor protein. In higher organisms including mammals, the insulin and IGF ligands and their receptors diverged and assumed more circumscribed functions. The contemporary view is that IGFs serve dominant roles in cell proliferation, survival and organismal growth, whereas insulin's primaryfunctionsinclude the regulation of fuel accumulation, storage and energy expenditure. However, such a simplistic modeltends to obscure the fact that insulin and IGF-1 continue to perform overlapping roles in several physiological processes. During the last funding period, we used mice lacking either IGF-1 or insulin receptor in osteoblasts to begin to distinguish actions of IGF-1 and insulin in the skeleton. These studies led to our discovery of two previously unappreciated roles for insulin signaling in bone. First, insulin suppresses the Runx2 inhibitor Twist2, which promotes osteoblast differentiation necessary for normal bone formation. Second, insulin induces production of osteocalcin (OC), which influences glucose utilization and energy expenditure. These results, which are described in the next section of the proposal, suggest the existence of a novel endocrine regulatory loop through which insulin signaling in the osteoblast controls postnatal bone development and stimulates OC production, which in turn, regulates pancreatic insulin secretion. The overall goal of this project will test the validity of this model in two putative effector cells: the osteoblast and pancreatic b-cell. The studiesare divided into two Specific Aims. Specific Aim 1: Characterize insulin actionsin the osteoblast. Studies during the last funding period indicate that insulin signaling in the osteoblasts is essential for normal osteoblast differentiation and postnatal bone accumulation but also directly stimulates OC production and g-carboxylation. In this aim, we will define the full range of insulin actions in osteoblasts. We will determine the mechanisms responsible for insulin transcriptional activation of OC production and posttranslational g-carboxylation. We will characterize the mechanisms through which insulin regulates glucose transport in the osteoblast and determine the role of the osteoblast glucose transporter-4 in global glucose utilization. Finally, to test the validity of putative bone-pancreas endocrine loop in vivo, we will determine the effect of a hyperinsulinemic-euglycemic clamp on circulating undercarboxylated OC levels in both wild-type and mice lacking Glut4 in osteoblasts. In essence, these studies will test the novel hypothesis that the osteoblast plays a critical role in the maintenance of glucose homeostasis. Specific Aim 2: Define the mechanisms through which OC influences pancreatic ss-cell function Preliminary studies suggest that OC increases pancreatic insulin secretion by increasing intracellular calcium ([Ca2+]i ), which we postulate occurs secondarily to interaction with a G-protein receptor on the b-cell. In this aim, we will perform in vitro and in vivo studies to identify the putative G-protein receptor for OC. We will characterize the effects of undercarboxylated OC on insulin secretion and test the significance of a candidate GPCR (GRPC6a) that is expressed in the pancreatic ss-cell. In parallel we will screen for novel GPCRs that respond to OC. These studies will be performed in collaboration with Bryan Roth and the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH-PDSP), to determine if OC can activate distinct peptidergic and non-peptidergic GPCRs. Candidates found in these screens will be interrogated by assessment of OC induced signals following ectopic expression in OC non- responsive cell types. An understanding of this pathway should ultimately enable better management of diabetic veterans and conceivably lead to the design a single therapy, which can simultaneously target both osteoporosis and type 2 diabetes.